Stapled acid-sensitive endosome disrupting alginates

ABSTRACT

An agent comprising:
         an algin crosslinked with acetal linkages; and   at least one cation coupled to the crosslinked algin.

This application is the U.S. National Stage of International ApplicationNo. PCT/US2016/039873, filed Jun. 28, 2016, which was published inEnglish under PCT Article 21(2), which in turn claims the benefit ofU.S. Provisional Application No. 62/186,141, filed Jun. 29, 2015, whichapplication is incorporated herein by reference in its entirety.

BACKGROUND

Cations such as calcium, copper, iron, zinc, and magnesium among othersare the most abundant minerals and are essential in several signalingpathways in all biological systems. Both eukaryotic and prokaryoticcells are efficient in maintaining optimum levels of minerals in thecytosol, and therefore, selectively increasing the intracellular levelsof minerals in order to modulate the cytosolic signaling pathways hasbeen challenging. For example, intracellular Cu to Zn ratio ismaintained and utilized by enzymes such as catalase to preventaccumulation of excess hydrogen peroxide, and superoxide dismutase toprevent excess accumulation of radical oxygen. Disrupting this mineralhomeostasis has been demonstrated to be effective in inducing cell deathselectively in cancer cells as compared to non-cancerous cells. Forexample, superoxide mimics, such as copper(II)(3,5-diisopropylsalicylate)₂ (Cu(II)DIPS) have been shown to inducehydrogen peroxide accumulation due to the conversion of radical oxygeninto hydrogen peroxide, with this accumulation of hydrogen peroxideleading to cell death in cancer cells. Conversely, given that relativelylow levels of superoxide are generated in normal cells, increasing thelevels of hydrogen peroxide in non-cancerous cells can lead to theirproliferation and survival. Currently, delivery of cell permeable Cu(II) requires the use of solvents such as dimethyl sulfoxide (DMSO) orethanol and therefore making the formulation not particularly suitablefor clinical translation. Therefore, there remains a need fordevelopment of effective delivery vehicles for cations, such as Cu (II),to the cytosol of the cells.

SUMMARY

Disclosed herein are endosome disrupting polymer alginoketals that canbe formulated into particles, and deliver any cation to the cytosol ofthe cells, thereby modulating their functions.

Disclosed herein is an agent comprising:

an algin crosslinked with acetal linkages; and

at least one cation coupled to the crosslinked algin.

Also disclosed herein is an agent comprising:

an algin monosaccharide residue covalently bonded to an acetal groupforming an alginoketal; and

at least one cation coupled to the alginoketal.

Further disclosed herein is an agent having a structure comprising:

wherein M is a cation; X¹ and X² are each independently —NH— or —O—, andA includes an acetal group;

wherein M is a cation; X¹ and X² are each independently —NH— or —O—, andA includes an acetal group;

or a combination of Formula IV and Formula V.

The foregoing will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Alginoketals deliver cations to the cytosol of the cells formodulating the cytosolic signaling pathways. Alginoketals have endosomedisrupting capability, and are held together, at least in part, bycations as the binding agent. Upon endocytosis, alginoketals disrupt theendosome and release cations in a sustained manner thereby modulatingintracellular signaling pathways.

FIGS. 2A-2C: Alginoketals were generated by crosslinking alginate andbis-amino ketals. A) The scheme of the reaction between alginic acid andbis-amino ketal. B) 1H NMR was obtained for the alginoketals and showsthe peaks of bis-amino ketal and alginate. C) The FTIR analysis showedthat the shift in the peak of carboxylic acid vibrations from 1595 inalginate to 1620 in alginoketals suggests the formation of an amidebond.

FIGS. 3A-3D: Size of the alginoketal particles generated using inverseemulsion. A) Alginoketal particles with Ca as the binding agent weregenerated and characterized using SEM and DLS, and there average sizewas 1 μm. B) Alginoketal particles with Cu as the binding agent weregenerated and characterized using SEM and DLS, and their size wasapproximately 0.8 μm. C) Alginoketal particles were incubated at pH 7.4for 6 days and the amount of OVA released was measured. The figure showsthat at pH 7.4 alginoketal particles release only 20% of theencapsulated OVA. D) Alginoketal particles were incubated at pH 5.6 for4 hours and the OVA released was quantified. At pH 5.6 Alginoketalparticles released 100% of the encapsulated OVA within 2 hr.

FIGS. 4A-4C: Alginoketal particles are endosome disruptors. A) M05 cellswere incubated with alginoketal-Ca particles and calcein for 15 min. Thefluorescence shown here in green in the cytosol of the cells,demonstrates that alginoketal particles disrupted the endosomes of M05and delivered calcein to the cytosol. B) M05 cells were incubated withalginate particles and calcein for 15 min. The absence of fluorescencein the cytosol of the cells, demonstrates that alginate particles couldnot disrupted the endosomes of M05 cells. C) M05 cells were incubatedwith calcein for 15 min. The absence of fluorescence in the cytosol ofthe cells, demonstrates that calcein by themselves do not reach thecytosol of the M05 cells.

FIGS. 5A-5H: Cell death due to hydrogen peroxide accumulation in M05melanoma cancer cell due to alginoketal-Cu. A,B) Alginoketal-Cuparticles scavenge radical oxygen, thereby preventing the oxidation ofTMB substrate, whereas alginoketal-Ca particles were not able toscavenge the radical oxygen. C,D) Alginoketal-Cu particles may act likesuperoxide dismutase and were able to generate hydrogen peroxide fromradical oxygen as determined by the oxidation of TMB in the presence ofhydrogen peroxide and horseradish peroxidase, whereas the negativecontrol Alginoketal-Ca particles did not induce hydrogen peroxideproduction. E) Alginoketal-Cu particles at 100 μg/mL induced higher celldeath in cancer cell types (M05 and HeLa) as compared to non-cancerouscell types (MSCs, HUVECs and HEK293). F) Addition of alginoketal-Cuparticles induce cell death in M05 melanoma cancer cells in a dosedependent manner, as determined by live/dead assay after 16 h ofincubation with Alginoketal-Cu or Alginoketal-Ca particles. G)Alginoketal-Cu particles induced intracellular production of hydrogenperoxide in the M05 cells in a dose-dependent manner, as observed by theoxidation of DCF, after 1 h of incubation. H) Qualitative observation ofalginoketal-Cu particles induced hydrogen peroxide in M05 cells asobserved by intracellular fluorescence due to hydrogen peroxideproduction, after 1 h of incubation with Alginoketal-Cu particles andM05 cells. i) 100 μg/mL Alginoketal-Cu particles ii) 50 μg/mLAlginoketal-Cu particles iii) 25 μg/mL Alginoketal-Cu particles iv) 10μg/mL Alginoketal-Cu particles v) 100 μg/mL Alginoketal-Ca particles vi)No treatment.

FIGS. 6A-6D. Stapled acid-sensitive endosome disrupting alginatesmodulate survival in mice and tumor growth. (FIG. 6A) Mice were injectedwith 0.5×10⁵B16F10-OVA expressing cells in the right and left flank ofmice (shown in blue), and when the tumors were palpable alginoketalparticles mixed with thermoresponsive gel as described in the Examplebelow were injected in the periphery of one of the tumors. (FIG. 6B)Kaplan Meier survival curves were obtained after treatment withparticles till day 28. Tumor growth was measured in the (FIG. 6C)ipsilateral and (FIG. 6D) contralateral side after treatment for 28 daysusing calipers. The tumor size for different treatment groups wasplotted against days and compared.

DETAILED DESCRIPTION Overview

Divalent cations, the most prevalent minerals in the body areresponsible for a wide variety of cellular functions includingsignaling, proliferation, differentiation and cell death, and thereforetheir transmembrane transportation is tightly regulated. Several of thedivalent cations such as Cu and Fe are toxic to the cells, andtherefore, their homeostasis is regulated through a system of membranebound protein transporters. For example, membrane transport protein Ctrlregulates the amount of Cu transported into the cells. Cations, such asCu and Zn are indispensable to the function of various cytosolicsignaling pathways and enzymes, and therefore, modulating their levelsin the cytosol is attractive. However, due to the tight regulation ofthese cations, increasing the intracellular levels of cations is achallenge. Despite the importance of divalent cations in cell activity,there are currently no intracellular delivery methods for divalentcations or modulation of intracellular levels of minerals.

Described herein are endosome disrupting alginate nanoparticles termed“alginoketals,” which can deliver cations to the cytosol of the cells.Alginoketals are generated by crosslinking alginic acid with endosomedisrupting ketals, and using cations as the stapling or binding agent.For example, alginoketals are able to deliver copper (II) in the cytosolof the cancer cells thereby disrupting copper homeostasis and inducingcell death via accumulation of hydrogen peroxide. Alginoketal-copper(II) based particles act as superoxide dismutase mimics and are thefirst class of divalent cation delivery vehicles, with potentialapplication in cancer therapy, regenerative medicine and drug delivery.

The polymers disclosed herein are excellent for delivering cations,because of their inherent capacity to chelate cations. Notably,alginoketal particles can be generated by intercalating cations withinan alginate matrix, and therefore, can capture, for example, Ca, Zn, Fe,Cu and Mg. Nanoparticles could be generated from these polymers using aninverse emulsion resulting in endocytosable delivery vehicles for Cu(II) and other cations. This is significant because, until now, deliveryof Cu (II), has required the use of hydrophobic chelators such asCu(II)DIPS, which (in turn) require organic solvents for delivery.Therefore, biodegradable alginoketal particles represent a new and moretranslatable organic solvent-free option for delivering cations such asCu (II).

The endosome disrupting ability of alginoketal polymers is demonstratedherein. Low pH-driven hydrolysis of the ketal or acetal linkages in thealginoketal particle can induce osmotic swelling of the endosomes andtherefore, lead to its disruption. Although, ketal linkages havepreviously been utilized to generate drug delivery vehicles, theprevious polymers cannot be utilized to deliver cations because of theirinability to chelate cations. On the other hand, alginoketal particlesare the only endosome disrupting polymers that can chelate cations, andtherefore, are an excellent resource for divalent cation delivery to thecytosol of the cells. We demonstrate that Cu (II) (which is a bindingagent of alginoketal particles) can be delivered to the cytosol of thecells. Once in the cytosol, alginoketal-Cu particles act as a superoxidedismutase mimic and can generate hydrogen peroxide from superoxides,which modulates cell function. Notably, superoxide dismutase mimics suchas Cu(II)DIPS have been shown to be effective in clearing tumors inmice. Accordingly, the in vitro experiments disclosed herein suggestthat alginoketal-Cu administration leads to M05 melanoma cell death.This can be attributed to the accumulation of hydrogen peroxide.Non-cancerous cells produce substantially less superoxide than cancercells, and therefore, it is expected that the accumulation of hydrogenperoxide, due to superoxide dismutase mimics, in non-cancerous cellswill not cause substantial cell death.

In certain embodiments:

-   -   Alginoketals can also be made positively charged and thereby        target the negatively charged surfaces such as cell surfaces and        biofilms among others without being cytotoxic;    -   The alginoketal is acid-sensitive, that is it degrades faster in        the presence of acid than in the presence of physiologic pH;    -   The alginoketal is the first class of particles that can deliver        cations such as, copper(II), iron(II), calcium(II) zinc (II)        among others.    -   The alginoketals can encapsulate an immunotherapeutic agent        and/or cancer cytotoxic agent.

Terminology

The following explanations of terms and methods are provided to betterdescribe the present compounds, compositions and methods, and to guidethose of ordinary skill in the art in the practice of the presentdisclosure. It is also to be understood that the terminology used in thedisclosure is for the purpose of describing particular embodiments andexamples only and is not intended to be limiting.

“Administration” as used herein is inclusive of administration byanother person to the subject or self-administration by the subject.

“Algins” as used herein includes alginic acid and salts or esters ofalginic acid, irrespective of the relative proportion of mannuronic andguluronic units, and is intended to include glycolated or alkoxylatedderivatives. Algins may be found in and isolated from various organisms,in particular from algae belonging to the order Phaeophyceae and soilbacteria such as Azotobacter vinelandii and Azotobacter crococcum andfrom several strains of Pseudomonas bacteria. Common algal sources ofalgins include Laminaria digitata, Ecklonia maxima, Macrocystispyrifera, Lessonia nigrescens, Ascophyllum nodosum, Laminariajaponica,Durvillea antartica, Durvillea potatorum and, especially, Laminariahyperborea. Alginic acid is a linear hetero-polysaccharide comprisingunits of β-D-mannuronic acid and α-L-guluronic acid. Alginic acid maycomprise homopolymeric sequences of mannuronic acid, homopolymericsequences of guluronic acid, and mixed sequences of mannuronic acid andguluronic acid units.

The term “aliphatic” is defined as including alkyl, alkenyl, alkynyl,halogenated alkyl and cycloalkyl groups. A “lower aliphatic” group is abranched or unbranched aliphatic group having from 1 to 10 carbon atoms.

“Alkanediyl” refers to a divalent radical derived from aliphatichydrocarbons.

The term “alkyl” refers to a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A“lower alkyl” group is a saturated branched or unbranched hydrocarbonhaving from 1 to 6 carbon atoms. Preferred alkyl groups have 1 to 4carbon atoms. Alkyl groups may be “substituted alkyls” wherein one ormore hydrogen atoms are substituted with a substituent such as halogen,cycloalkyl, alkoxy, amino, hydroxyl, aryl, alkenyl, or carboxyl. Forexample, a lower alkyl or (C₁-C₆)alkyl can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;(C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl; (C₃-C₆)cycloalkyl(C₁-C₆)alkyl can be cyclopropylmethyl,cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl,2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or2-cyclohexylethyl; (C₁-C₆)alkoxy can be methoxy, ethoxy, propoxy,isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, orhexyloxy; (C₂-C₆)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl,4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl;(C₂-C₆)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl,1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl;(C₁-C₆)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C₁-C₆)alkylcan be iodomethyl, bromomethyl, chloromethyl, fluoromethyl,trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, orpentafluoroethyl; hydroxy(C₁-C₆)alkyl can be hydroxymethyl,1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl,3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl,5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl;(C₁-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl; (C₁-C₆)alkylthio can be methylthio, ethylthio,propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, orhexylthio; (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy,isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

An “animal” refers to living multi-cellular vertebrate organisms, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and non-human subjects, including birds andnon-human mammals, such as non-human primates, companion animals (suchas dogs and cats), livestock (such as pigs, sheep, cows), as well asnon-domesticated animals, such as the big cats. The term subject appliesregardless of the stage in the organism's life-cycle. Thus, the termsubject applies to an organism in utero or in ovo, depending on theorganism (that is, whether the organism is a mammal or a bird, such as adomesticated or wild fowl).

“Inhibiting” refers to inhibiting the full development of a disease orcondition. “Inhibiting” also refers to any quantitative or qualitativereduction in biological activity or binding, relative to a control.

A “therapeutically effective amount” refers to a quantity of a specifiedagent sufficient to achieve a desired effect in a subject being treatedwith that agent. Ideally, a therapeutically effective amount of an agentis an amount sufficient to inhibit or treat the disease or conditionwithout causing a substantial cytotoxic effect in the subject. Thetherapeutically effective amount of an agent will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration of the therapeutic composition.

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop, or administering a compound or composition to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing a pathology or condition,or diminishing the severity of a pathology or condition. As used herein,the term “ameliorating,” with reference to a disease or pathologicalcondition, refers to any observable beneficial effect of the treatment.The beneficial effect can be evidenced, for example, by a delayed onsetof clinical symptoms of the disease in a susceptible subject, areduction in severity of some or all clinical symptoms of the disease, aslower progression of the disease, an improvement in the overall healthor well-being of the subject, or by other parameters well known in theart that are specific to the particular disease. The phrase “treating adisease” refers to inhibiting the full development of a disease, forexample, in a subject who is at risk for a disease such as cancer.“Preventing” a disease or condition refers to prophylactic administeringa composition to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping a pathology or condition, or diminishing the severity of apathology or condition.

“Pharmaceutical compositions” are compositions that include an amount(for example, a unit dosage) of one or more of the disclosed compoundstogether with one or more non-toxic pharmaceutically acceptableadditives, including carriers, diluents, and/or adjuvants, andoptionally other biologically active ingredients. Such pharmaceuticalcompositions can be prepared by standard pharmaceutical formulationtechniques such as those disclosed in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. (19th Edition).

The terms “pharmaceutically acceptable salt or ester” refers to salts oresters prepared by conventional means that include salts, e.g., ofinorganic and organic acids, including but not limited to hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonicacid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid,tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid,maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelicacid and the like. “Pharmaceutically acceptable salts” of the presentlydisclosed compounds also include those formed from cations such assodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and frombases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)aminomethane, andtetramethylammonium hydroxide. These salts may be prepared by standardprocedures, for example by reacting the free acid with a suitableorganic or inorganic base. Any chemical compound recited in thisspecification may alternatively be administered as a pharmaceuticallyacceptable salt thereof. “Pharmaceutically acceptable salts” are alsoinclusive of the free acid, base, and zwitterionic forms. Descriptionsof suitable pharmaceutically acceptable salts can be found in Handbookof Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH(2002). When compounds disclosed herein include an acidic function suchas a carboxy group, then suitable pharmaceutically acceptable cationpairs for the carboxy group are well known to those skilled in the artand include alkaline, alkaline earth, ammonium, quaternary ammoniumcations and the like. Such salts are known to those of skill in the art.For additional examples of “pharmacologically acceptable salts,” seeBerge et al., J. Pharm. Sci. 66:1 (1977).

“Pharmaceutically acceptable esters” includes those derived fromcompounds described herein that are modified to include a carboxylgroup. An in vivo hydrolysable ester is an ester, which is hydrolysed inthe human or animal body to produce the parent acid or alcohol.Representative esters thus include carboxylic acid esters in which thenon-carbonyl moiety of the carboxylic acid portion of the ester groupingis selected from straight or branched chain alkyl (for example, methyl,n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example,methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example,phenoxymethyl), aryl (for example, phenyl, optionally substituted by,for example, halogen, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy) or amino);sulphonate esters, such as alkyl- or aralkylsulphonyl (for example,methanesulphonyl); or amino acid esters (for example, L-valyl orL-isoleucyl). A “pharmaceutically acceptable ester” also includesinorganic esters such as mono-, di-, or tri-phosphate esters. In suchesters, unless otherwise specified, any alkyl moiety presentadvantageously contains from 1 to 18 carbon atoms, particularly from 1to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Anycycloalkyl moiety present in such esters advantageously contains from 3to 6 carbon atoms. Any aryl moiety present in such esters advantageouslycomprises a phenyl group, optionally substituted as shown in thedefinition of carbocycylyl above. Pharmaceutically acceptable estersthus include C₁-C₂₂ fatty acid esters, such as acetyl, t-butyl or longchain straight or branched unsaturated or omega-6 monounsaturated fattyacids such as palmoyl, stearoyl and the like. Alternative aryl orheteroaryl esters include benzoyl, pyridylmethyloyl and the like any ofwhich may be substituted, as defined in carbocyclyl above. Additionalpharmaceutically acceptable esters include aliphatic L-amino acid esterssuch as leucyl, isoleucyl and especially valyl.

For therapeutic use, salts of the compounds are those wherein thecounter-ion is pharmaceutically acceptable. However, salts of acids andbases which are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound.

The pharmaceutically acceptable acid and base addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid and base addition salt forms which the compounds are ableto form. The pharmaceutically acceptable acid addition salts canconveniently be obtained by treating the base form with such appropriateacid. Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds containing an acidic proton may also be converted intotheir non-toxic metal or amine addition salt forms by treatment withappropriate organic and inorganic bases. Appropriate base salt formscomprise, for example, the ammonium salts, the alkali and earth alkalinemetal salts, e.g. the lithium, sodium, potassium, magnesium, calciumsalts and the like, salts with organic bases, e.g. the benzathine,N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids suchas, for example, arginine, lysine and the like.

The term “addition salt” as used hereinabove also comprises the solvateswhich the compounds described herein are able to form. Such solvates arefor example hydrates, alcoholates and the like.

The term “quaternary amine” as used hereinbefore defines the quaternaryammonium salts which the compounds are able to form by reaction betweena basic nitrogen of a compound and an appropriate quaternizing agent,such as, for example, an optionally substituted alkylhalide, arylhalideor arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactantswith good leaving groups may also be used, such as alkyltrifluoromethanesulfonates, alkyl methanesulfonates, and alkylp-toluenesulfonates. A quaternary amine has a positively chargednitrogen. Pharmaceutically acceptable counterions include chloro, bromo,iodo, trifluoroacetate and acetate. The counterion of choice can beintroduced using ion exchange resins.

Prodrugs of the disclosed compounds also are contemplated herein. Aprodrug is an active or inactive compound that is modified chemicallythrough in vivo physiological action, such as hydrolysis, metabolism andthe like, into an active compound following administration of theprodrug to a subject. The term “prodrug” as used throughout this textmeans the pharmacologically acceptable derivatives such as esters,amides and phosphates, such that the resulting in vivo biotransformationproduct of the derivative is the active drug as defined in the compoundsdescribed herein. Prodrugs preferably have excellent aqueous solubility,increased bioavailability and are readily metabolized into the activeinhibitors in vivo. Prodrugs of a compounds described herein may beprepared by modifying functional groups present in the compound in sucha way that the modifications are cleaved, either by routine manipulationor in vivo, to the parent compound. The suitability and techniquesinvolved in making and using prodrugs are well known by those skilled inthe art. F or a general discussion of prodrugs involving esters seeSvensson and Tunek, Drug Metabolism Reviews 165 (1988) and Bundgaard,Design of Prodrugs, Elsevier (1985).

The term “prodrug” also is intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when the prodrug is administered to a subject. Since prodrugs oftenhave enhanced properties relative to the active agent pharmaceutical,such as, solubility and bioavailability, the compounds disclosed hereincan be delivered in prodrug form. Thus, also contemplated are prodrugsof the presently disclosed compounds, methods of delivering prodrugs andcompositions containing such prodrugs. Prodrugs of the disclosedcompounds typically are prepared by modifying one or more functionalgroups present in the compound in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to yield the parentcompound. Prodrugs include compounds having a phosphonate and/or aminogroup functionalized with any group that is cleaved in vivo to yield thecorresponding amino and/or phosphonate group, respectively. Examples ofprodrugs include, without limitation, compounds having an acylated aminogroup and/or a phosphonate ester or phosphonate amide group. Inparticular examples, a prodrug is a lower alkyl phosphonate ester, suchas an isopropyl phosphonate ester.

Protected derivatives of the disclosed compounds also are contemplated.A variety of suitable protecting groups for use with the disclosedcompounds are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

In general, protecting groups are removed under conditions that will notaffect the remaining portion of the molecule. These methods are wellknown in the art and include acid hydrolysis, hydrogenolysis and thelike. One preferred method involves the removal of an ester, such ascleavage of a phosphonate ester using Lewis acidic conditions, such asin TMS-Br mediated ester cleavage to yield the free phosphonate. Asecond preferred method involves removal of a protecting group, such asremoval of a benzyl group by hydrogenolysis utilizing palladium oncarbon in a suitable solvent system such as an alcohol, acetic acid, andthe like or mixtures thereof. A t-butoxy-based group, including t-butoxycarbonyl protecting groups can be removed utilizing an inorganic ororganic acid, such as HCl or trifluoroacetic acid, in a suitable solventsystem, such as water, dioxane and/or methylene chloride. Anotherexemplary protecting group, suitable for protecting amino and hydroxyfunctions amino is trityl. Other conventional protecting groups areknown and suitable protecting groups can be selected by those of skillin the art in consultation with Greene and Wuts, Protective Groups inOrganic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999. When anamine is deprotected, the resulting salt can readily be neutralized toyield the free amine. Similarly, when an acid moiety, such as aphosphonic acid moiety is unveiled, the compound may be isolated as theacid compound or as a salt thereof.

Particular examples of the presently disclosed agents may include one ormore asymmetric centers; thus these compounds can exist in differentstereoisomeric forms. Accordingly, compounds and compositions may beprovided as individual pure enantiomers or as stereoisomeric mixtures,including racemic mixtures. In certain embodiments the compoundsdisclosed herein are synthesized in or are purified to be insubstantially enantiopure form, such as in a 90% enantiomeric excess, a95% enantiomeric excess, a 97% enantiomeric excess or even in greaterthan a 99% enantiomeric excess, such as in enantiopure form.

The presently disclosed agents can have at least one asymmetric centeror geometric center, cis-trans center (C═C, C═N). All chiral,diasteromeric, racemic, meso, rotational and geometric isomers of thestructures are intended unless otherwise specified. The compounds can beisolated as a single isomer or as mixture of isomers. All tautomers ofthe compounds are also considered part of the disclosure. The presentlydisclosed compounds also includes all isotopes of atoms present in thecompounds, which can include, but are not limited to, deuterium,tritium, ¹⁸F, etc.

Alginoketals and Alginoketal-M Agents

Disclosed herein are polymers, agents, compositions and methods fortransporting or releasing therapeutic and diagnostic agents ormetabolites or other analytes from cells, compartments within cells,through cell layers or cell barriers, or lipid membranes. The polymers,agents or compositions are endosomal disruptive materials that becomemembrane disruptive following endocytosis, releasing a therapeutic,diagnostic or prophylactic agent for delivery.

The endosomal disruptive materials are disrupted upon exposure to anappropriate stimulus, typically a change in pH, and most typically adecrease in pH from physiological pH (i.e., typically pH 7.4) to the pHof the endosome (approximately between 5 and 6.5). The endosomaldisruptive materials include an acid labile acetal linkage which iscleaved following or during endocytosis.

The alginoketal includes an algin component(s) directly linked to aketal component(s). The direct linkage preferably is via a covalentbond. In certain embodiments, the ketal component(s) forms an acetallinkage between algin polymer chains or within the same algin polymerchain. The acetal linking group may crosslink and/or chain extend thealgin polymer chains. In certain embodiments, the acetal linking groupis bonded to a carboxyl group present on an algin monosaccharideresidue. In certain embodiments, the algin is directly conjugated to theketal. In other words, there are no spacers or any other components inthe alginoketal backbone structure. Examples of alginoketal structuresare shown in FIGS. 1 and 2 and below:

wherein X¹ and X² are each independently —NH— or —O—, and A includes anacetal group. More particularly, A is

wherein R¹⁰ and R¹¹ are each independently an alkanediyl, and R¹² andR¹³ are each optionally substituted alkyl.

In certain embodiments, X¹ and X² are the same.

In certain embodiments, R¹⁰ and R¹¹ are the same, and are particularly(C₁-C₆)alkanediyl.

In a further embodiment, the alginoketal includes a structure of:

wherein Y¹ is an alkanediyl, R² is optionally substituted alkyl, andeach R³ is independently optionally substituted alkyl or an alginmonosaccharide residue. In certain embodiments, at least one R³ is analgin monosaccharide residue.

In a specific embodiment, the alginoketal includes a structure of:

Alginic acid itself may be used the presently disclosed methods as astarting material for making the alginoketals. However, salts of alginicacid may also be used in the methods and may include alkali metal salts,for example sodium and potassium salts, and ammonium and alkanolaminesalts. Esters of alginic acid may also be used in the methods providedthere is a sufficient number of free carboxyl groups available forconjugating with the ketal. The algin starting material is the precursorfor the algin monosaccharide residues present in the alginoketal.

The ketal used to make the alginoketal may be any compound that includesat least one ketal group and at least one functional group that isreactive with a carboxyl group or a hydroxyl group present on an alginmonosaccharide residue. In certain embodiments, the ketal includes anamine group that is reactive with the carboxyl group thus generating anamide linkage. In certain embodiments, the ketal includes a hydroxylgroup that is reactive with the carboxyl group thus generating an esterlinkage. In certain embodiments, the ketal includes a halogen group thatis reactive with the hydroxyl group of the algin monosaccharide residue.Illustrative ketals include 2,2-bis(aminoethoxy)propane,1-(2-Hydroxyethyl)-4-piperidone ethylene ketal,1-Amino-4-oxocyclohexanecarboxylic acid ethylene ketal, and4-Bromoacetophenone diethyl ketal.

In certain embodiments, a ketone could be introduced onto the alginpolymer, and then converted to a ketal.

A divalent cation may be coupled to the alginoketal. In certainembodiments, the cation may be a metal cation or non-metal cation.Illustrative cations include copper (II), iron (II), calcium (II), zinc(II), magnesium (II), selenium, aluminium, manganese, barium, andstrontium. A cation-bearing compound is contacted with the alginoketalunder conditions for coupling the cation with the alginoketal. Thecation-bearing compound may be a salt of the above-identified divalentcations. Illustrative salts include sulfate, phosphate, chloride,bromide, fluoride, and pyrophosphate.

Certain embodiments disclosed herein are the first class of polymersthat can deliver several different kinds of cations simultaneously. Inother words, a single molecule of alginoketal can be loaded with morethan one type of cation (e.g., Cu(II) and Ca(II)). The amount of cationsper alginoketal may vary. For example, the cation content may range from1 picomole to 1 mole, more particularly 1 nanomole to 1 micromole, andmost particularly 1 nanomole to 100 nanomole.

In certain embodiments, the cation is chelated to the alginoketal. Forexample, in certain embodiments, the cation is covalently or ionicbonded to a carboxyl and/or hydroxyl group present on the alginoketal.Illustrative examples of alginoketal-cation structures are shown in areshown in FIG. 1 and below.

For example, the alginoketal-cation may include a structure of:

wherein M is a cation; X¹ and X² are each independently —NH— or —O—, andA includes an acetal group. More particularly, A is

wherein R¹⁰ and R¹¹ are each independently an alkanediyl, and R¹² andR¹³ are each optionally substituted alkyl.

In certain embodiments, X¹ and X² are the same.

In certain embodiments, R¹⁰ and R¹¹ are the same, and are particularly(C₁-C₆)alkanediyl.

In a further example, the alginoketal-cation may include a structure of:

wherein M is a cation; X¹ and X² are each independently —NH— or —O—, andA includes an acetal group. More particularly, A is

wherein R¹⁰ and R¹¹ are each independently an alkanediyl, and R¹² andR¹³ are each optionally substituted alkyl.

In certain embodiments, X¹ and X² are the same.

In certain embodiments, R¹⁰ and R¹¹ are the same, and are particularly(C₁-C₆)alkanediyl. In a further example, the alginoketal-cation mayinclude a structure of:

wherein M is a cation, Y¹ is an alkanediyl, R² is optionally substitutedalkyl, and each R³ is independently optionally substituted alkyl or analgin monosaccharide residue, provided that at least one of R³ is analgin monosaccharide residue.

In a further example, the alginoketal-cation may include a structure of:

wherein M is a divalent cation, Y¹ is an alkanediyl, R² is optionallysubstituted alkyl, and each R³ is independently optionally substitutedalkyl or an algin monosaccharide residue, provided that at least one ofR³ is an algin monosaccharide residue.

In a further example, the alginoketal-cation may include a structure of:

wherein M is a divalent cation, Y¹ is an alkanediyl, R² is optionallysubstituted alkyl, and each R³ is independently optionally substitutedalkyl or an algin monosaccharide residue, provided that at least one ofR³ is an algin monosaccharide residue.

In a further example, the alginoketal-cation may include a structure of:

wherein M is a divalent cation, Y¹ is an alkanediyl, R² is optionallysubstituted alkyl, and each R³ is independently optionally substitutedalkyl or an algin monosaccharide residue, provided that at least one ofR³ is an algin monosaccharide residue.

The alginoketal is generated in a single step from a naturally-occurringpolymer (alginic acid) that is easily obtainable. For example, thealginoketal may be made by forming an alginic acid solution in DI water.1 to 100 fold stoichiometric excess of 2,2-Bis(aminoethoxy)propane oranother compound that contains ketal linkages are added to the alginicacid solution with 100-500 fold stoichiometric excess of crosslinkingagents (e.g., sulfo-NHS and EDC)), which leads to the formation of anamide bond. Other crosslinking or catalytic agents leading to theformation of amide are 5-methoxy-2-iodophenylboronic acid (MIBA),XtalFluor-E, Nanosized sulfated titania, T3P (n-propanephosphonic acidanhydride), HBTU, prop-2-ene-1-sulfinyl+DMAP, B(OCH2CF3)3,trimethylaluminium, 3,3-dichlorocyclopropenes among others. The reactionis continued for 24-48 hours at 25-37 degrees C. under stirring. Thereaction product is then dialyzed using a filter 500-10000 Da molecularweight cut-off to remove the un-reacted reaction components.

The alginoketal-cation agent may be in any form. For example, cationsmay be bound to alginoketals by adding a 1-1000 mM solution of cationsalt to the 1-100 mL solution of 0.1-10% of alginoketal solution understirring. The resulting alginoketal-cation polymer may be used asdesired. For example, the alginoketal-cation polymer may be coated on asurface to form a hydrogel. Applications may include making the coatedsurface bacteria resistant, and encapsulating cells.

In certain embodiments, the alginoketal-cation agent is formulated asparticle. The alginoketal-cation particles may have any dimension, buttypically are microparticles or nanoparticles. For example, themicroparticles may have an average size of 1-999 μm or less than 999 μm,more particularly less than 500 μm, and most particularly less than 100μm. In certain embodiments, the nanoparticles may have an average size1-999 nm of less than 999 nm, more particularly less than 500 nm, andmost particularly less than 250 nm.

The alginoketal-cation particles may be generated by any suitablemethod. For example, the particles may be generated via a reverseemulsion process. In particular, a 0.1-10% solution of alginoketals inDI H2O may be generated and used as the water phase. Span 80 (0.1-10 mL)(or another surfactant or mixture of surfactants) can be dissolved iniso-octane (10-1000 mL), and used as the oil phase (other oil phasessuch as toluene can be used). In order to generate protein encapsulatedalginoketal particles, the desired protein (0.001-1000 mg) can be addedto the alginoketal solution and vortexed further for thorough mixing.This resulting solution of alginoketals, either containing protein ornot containing any protein, may then be added to the oil phase understirring and allowed to mix. DI H₂O or a cation solution (1-1000 mM)made in DI H₂O can then be added under stirring and allowed to mix.2-propanol (1-1000 mL) may then be added to the mixture in order to curethe particles, and allowed to mix. The particles obtained may becentrifuged and the supernatant was discarded. The particles can then bere-suspended in 2-propanol, incubated at room temperature and thencentrifuged again. The particles can then be re-suspended in 1×phosphate buffered saline and then centrifuged again.

In certain embodiments, the alginoketal can encapsulate animmunotherapeutic agent and/or cancer cytotoxic agent.

In certain embodiments, a protein is encapsulated within the alginoketalIllustrative immunotherapeutic agents for encapsulation includeanti-PD-1, anti-PD-L1, anti-CTLA4, anti-Tim3, anti-Lag3, anti-OX40,IL-2, IFN gamma, IFN alpha, anti-CCL22, MPLA, poly(I:C), and CpG.

Illustrative cytotoxic agents for encapsulation include 1-MT.

Alginoketal particles can be produced by different techniques includingbut not limited to air atomization, emulsification (as described in thisembodiment), complexation with counterion polymers (such as chitosan,poly-1-lysine, polyethylene imine among others), spray-drying,electrohydrodynamic atomization, impinging aerosols, precipitation, andinkjet/drying processes.

Uses

In certain embodiments, the alginoketal-M agents disclosed herein may beused for treating or inhibiting a neoplasm. For example, the agents maykill or otherwise inhibit cancer cells, by modulating the intracellularreduction-oxidation potential in the cancer cells, without significantlymodifying the potential in non-cancerous cells.

The agents and compositions disclosed herein may be useful for treatingany type of neoplasm (e.g., cancer). Tumors or neoplasms include newgrowths of tissue in which the multiplication of cells is uncontrolledand progressive. Some such growths are benign, but others are termed“malignant,” leading to death of the organism. Malignant neoplasms or“cancers” are distinguished from benign growths in that, in addition toexhibiting aggressive cellular proliferation, they invade surroundingtissues and metastasize. Moreover, malignant neoplasms are characterizedin that they show a greater loss of differentiation (greater“dedifferentiation”), and of their organization relative to one anotherand their surrounding tissues. This property is also called “anaplasia.”

Illustrative neoplasms include fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, multiple myeloma, and lymphoma.

In certain embodiments, the presently disclosed methods are directed toa method for inhibiting cancer growth, including processes of cellularproliferation, invasiveness, and metastasis in biological systems.Preferably, the method is employed to inhibit or reduce cancer cellproliferation, invasiveness, metastasis, or tumor incidence in livinganimals, such as mammals.

Also provided herein is a method of inducing cytotoxicity (cell killing)in cancer cells or reducing the viability of cancer cells. For example,the methods can be used to induce cytotoxicity in cells of carcinomas ofthe prostate, breast, ovary, testis, lung, colon, or pancreas.

The killing of cancer cells can occur with less cytotoxicity to normalcells or tissues than is found with conventional cytotoxic therapeutics,preferably without substantial cytotoxicity to normal cells or tissues.For example, the methods identified herein can induce cytotoxicity incancer cells while producing little or substantially no cytotoxicity innormal cells. Thus, unlike conventional cytotoxic anticancertherapeutics, which typically kill all growing cells, the methods canproduce differential cytotoxicity: tumor cells are selectively killedwhereas normal cells are spared. Thus, in another embodiment, there isdisclosed a method for inducing differential cytotoxicity in cancercells relative to normal cells or tissue.

In certain embodiments, the alginoketal-M agents disclosed herein may beused for targeting biofilms, and clearing bacteria such as oralbiofilms, and biomaterial induced biofilms (heart valves, hip and kneereplacements), among others.

In certain embodiments, the alginoketal-M agents disclosed herein may beused for delivery of divalent cations to the brain (e.g., via directinjection into the brain) for brain-related neurodegenerative disorderssuch as, for example, Alzheimer's disease, ataxia telangiectasia,Parkinson's disease, amyotrophic lateral sclerosis, and Huntington'sdisease.

In some embodiments, the methods disclosed herein involve administeringto a subject in need of treatment a therapeutically effective amount ofone or more of the agents disclosed herein. In some embodiments, themethods disclosed herein involve administering to a subject in need oftreatment a pharmaceutical composition, for example a composition thatincludes a pharmaceutically acceptable additive and a therapeuticallyeffective amount of one or more of the agents disclosed herein.

The agents may be administered orally, parenterally (includingsubcutaneous injections (SC or depo-SC), intravenous (IV), intramuscular(IM or depo-IM), intrasternal injection or infusion techniques),sublingually, intranasally (inhalation), intrathecally, topically,ophthalmically, rectally, intradermally or intraperitoneally. Thepharmaceutical composition may be administered in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants, and/or vehicles. The agents arepreferably formulated into suitable pharmaceutical preparations such astablets, capsules, or elixirs for oral administration or in sterilesolutions or suspensions for parenteral administration. Typically theagents described above are formulated into pharmaceutical compositionsusing techniques and procedures well known in the art.

In some embodiments, one or more of the disclosed agents are mixed orcombined with a suitable pharmaceutically acceptable carrier to preparea pharmaceutical composition. Pharmaceutical carriers or vehiclessuitable for administration of the agents provided herein include anysuch carriers known to be suitable for the particular mode ofadministration. Remington: The Science and Practice of Pharmacy, TheUniversity of the Sciences in Philadelphia, Editor, Lippincott,Williams, & Wilkins, Philadelphia, Pa., 21^(st) Edition (2005),describes exemplary compositions and formulations suitable forpharmaceutical delivery of the agents disclosed herein. In addition, thecompounds may be formulated as the sole pharmaceutically activeingredient in the composition or may be combined with other activeingredients.

Upon mixing or addition of the agent(s) to a pharmaceutically acceptablecarrier, the resulting mixture may be a solution, suspension, emulsion,or the like. Liposomal suspensions may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. Where the agents exhibit insufficient solubility,methods for solubilizing may be used. Such methods are known andinclude, but are not limited to, using cosolvents such asdimethylsulfoxide (DMSO), using surfactants such as Tween®, anddissolution in aqueous sodium bicarbonate. Derivatives of the agents,such as salts or prodrugs may also be used in formulating effectivepharmaceutical compositions. The disclosed agents may also be preparedwith carriers that protect them against rapid elimination from the body,such as time-release formulations or coatings. Such carriers includecontrolled release formulations, such as, but not limited to,microencapsulated delivery systems.

The disclosed agents and/or compositions can be enclosed in multiple orsingle dose containers. The agents and/or compositions can also beprovided in kits, for example, including component parts that can beassembled for use. For example, one or more of the disclosed agents maybe provided in a lyophilized form and a suitable diluent may be providedas separated components for combination prior to use. In some examples,a kit may include a disclosed agent and a second therapeutic agent forco-administration. The compound and second therapeutic agent may beprovided as separate component parts. A kit may include a plurality ofcontainers, each container holding one or more unit dose of the agent.The containers are preferably adapted for the desired mode ofadministration, including, but not limited to tablets, gel capsules,sustained-release capsules, and the like for oral administration; depotproducts, pre-filled syringes, ampoules, vials, and the like forparenteral administration; and patches, medipads, creams, and the likefor topical administration.

The active agent is included in the pharmaceutically acceptable carrierin an amount sufficient to exert a therapeutically useful effect in theabsence of undesirable side effects on the subject treated. Atherapeutically effective concentration may be determined empirically bytesting the agents in known in vitro and in vivo model systems for thetreated disorder. In some examples, a therapeutically effective amountof the agent is an amount that lessens or ameliorates at least onesymptom of the disorder for which the compound is administered.Typically, the compositions are formulated for single dosageadministration. The concentration of active agent in the drugcomposition will depend on absorption, inactivation, and excretion ratesof the active compound, the dosage schedule, and amount administered aswell as other factors known to those of skill in the art.

In some examples, about 0.1 mg to 1000 mg of a disclosed agent, amixture of such agents, or a physiologically acceptable salt or esterthereof, is compounded with a physiologically acceptable additive suchas an excipient, binder, preservative, stabilizer, flavor, etc., in aunit dosage form. The amount of active substance in those compositionsor preparations is such that a suitable dosage in the range indicated isobtained. The term “unit dosage form” refers to physically discreteunits suitable as unitary dosages for human subjects and other mammals,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect, in associationwith a suitable pharmaceutical excipient. In some examples, thecompositions are formulated in a unit dosage form, each dosagecontaining from about 1 mg to about 1000 mg (for example, about 2 mg toabout 500 mg, about 5 mg to 50 mg, about 10 mg to 100 mg, or about 25 mgto 75 mg) of the one or more agents. In other examples, the unit dosageform includes about 0.1 mg, about 1 mg, about 5 mg, about 10 mg, about20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg,about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg,about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg,about 700 mg, about 800 mg, about 900 mg, about 1000 mg, or more of thedisclosed agent(s).

The disclosed agents or compositions may be administered as a singledose, or may be divided into a number of smaller doses to beadministered at intervals of time. The therapeutic compositions can beadministered in a single dose delivery, by continuous delivery over anextended time period, in a repeated administration protocol (forexample, by a multi-daily, daily, weekly, or monthly repeatedadministration protocol). It is understood that the precise dosage,timing, and duration of treatment is a function of the disease beingtreated and may be determined empirically using known testing protocolsor by extrapolation from in vivo or in vitro test data. It is to benoted that concentrations and dosage values may also vary with theseverity of the condition to be alleviated. In addition, it isunderstood that for a specific subject, dosage regimens may be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only.

When administered orally as a suspension, these compositions areprepared according to techniques well known in the art of pharmaceuticalformulation and may contain microcrystalline cellulose for impartingbulk methylcellulose as a viscosity enhancer, and sweeteners/flavoringagents. As immediate release tablets, these compositions may containmicrocrystalline cellulose, dicalcium phosphate, starch, magnesiumstearate and lactose and/or other excipients, binders, extenders,disintegrants, diluents and lubricants. If oral administration isdesired, the agent is typically provided in a composition that protectsit from the acidic environment of the stomach. For example, thecomposition can be formulated in an enteric coating that maintains itsintegrity in the stomach and releases the active agent in the intestine.The composition may also be formulated in combination with an antacid orother such ingredient.

Oral compositions will generally include an inert diluent or an ediblecarrier and may be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, the activeagent can be incorporated with excipients and used in the form oftablets, capsules, or troches. Pharmaceutically compatible bindingagents and adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches, and the like cancontain any of the following ingredients or compounds of a similarnature: a binder such as, but not limited to, gum tragacanth, acacia,corn starch, or gelatin; an excipient such as microcrystallinecellulose, starch, or lactose; a disintegrating agent such as, but notlimited to, alginic acid and corn starch; a lubricant such as, but notlimited to, magnesium stearate; a gildant, such as, but not limited to,colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; and a flavoring agent such as peppermint, methyl salicylate,or fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials, whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The agents can also be administered as acomponent of an elixir, suspension, syrup, wafer, chewing gum or thelike. A syrup may contain, in addition to the active compounds, sucroseas a sweetening agent and certain preservatives, dyes and colorings, andflavors.

When administered orally, the agents can be administered in usual dosageforms for oral administration. These dosage forms include the usualsolid unit dosage forms of tablets and capsules as well as liquid dosageforms such as solutions, suspensions, and elixirs. For example, thedosage unit form may be an injectable fluid, an oral delivery fluid(e.g., a solution or suspension), a nasal delivery fluid (e.g., fordelivery as an aerosol or vapor), a semisolid form (e.g., a topicalcream), or a solid form such as powder, pill, tablet, or capsule forms.

When the solid dosage forms are used, it is preferred that they be ofthe sustained release type so that the compounds need to be administeredonly once or twice daily. In some examples, an oral dosage form isadministered to the subject 1, 2, 3, 4, or more times daily. In certainexamples, the oral dosage is from about 1 mg/day to about 500 mg/day,about 2 mg/day to about 200 mg/day, or about 5 mg/day to about 50mg/day. It is understood that while a subject may be started at onedose, that dose may be varied over time as the subject's conditionchanges.

In additional examples, the agents can be administered orally to humansin a dosage range of 1 to 1000 mg/kg body weight in single or divideddoses. One illustrative dosage range is 0.1 to 200 mg/kg body weightorally (such as 0.5 to 100 mg/kg body weight orally) in single ordivided doses. For oral administration, the compositions may be providedin the form of tablets containing about 1 to 1000 milligrams of theactive ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 75, 100, 150,200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 milligrams of theactive ingredient. It will be understood, however, that the specificdose level and frequency of dosage for any particular patient may bevaried and will depend upon a variety of factors including the activityof the specific agent employed, the metabolic stability and length ofaction of that compound, the age, body weight, general health, sex,diet, mode and time of administration, rate of excretion, drugcombination, the severity of the particular condition, and the hostundergoing therapy.

Injectable solutions or suspensions may also be formulated, usingsuitable non-toxic, parenterally-acceptable diluents or solvents, suchas mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodiumchloride solution, or suitable dispersing or wetting and suspendingagents, such as sterile, bland, fixed oils, including synthetic mono- ordiglycerides, and fatty acids, including oleic acid. Solutions orsuspensions used for parenteral, intradermal, subcutaneous, or topicalapplication can include any of the following components: a sterilediluent such as water for injection, saline solution, fixed oil, anaturally occurring vegetable oil such as sesame oil, coconut oil,peanut oil, cottonseed oil, and the like, or a synthetic fatty vehiclesuch as ethyl oleate, and the like, polyethylene glycol, glycerine,propylene glycol, or other synthetic solvent; antimicrobial agents suchas benzyl alcohol and methyl parabens; antioxidants such as ascorbicacid and sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid (EDTA); buffers such as acetates,citrates, and phosphates; and agents for the adjustment of tonicity suchas sodium chloride and dextrose. Parenteral preparations can be enclosedin ampoules, disposable syringes, or multiple dose vials made of glass,plastic, or other suitable material. Buffers, preservatives,antioxidants, and the like can be incorporated as required.

Where administered intravenously, suitable carriers includephysiological saline, phosphate buffered saline (PBS), and solutionscontaining thickening and solubilizing agents such as glucose,polyethylene glycol, polypropyleneglycol, and mixtures thereof.Liposomal suspensions including tissue-targeted liposomes may also besuitable as pharmaceutically acceptable carriers.

The agents can be administered parenterally, for example, by IV, IM,depo-IM, SC, or depo-SC. When administered parenterally, atherapeutically effective amount of about 0.1 to about 500 mg/day (suchas about 1 mg/day to about 100 mg/day, or about 5 mg/day to about 50mg/day) may be delivered. When a depot formulation is used for injectiononce a month or once every two weeks, the dose may be about 0.1 mg/dayto about 100 mg/day, or a monthly dose of from about 3 mg to about 3000mg.

The agents can also be administered sublingually. When givensublingually, the agents should be given one to four times daily in theamounts described above for IM administration.

The agents can also be administered intranasally. When given by thisroute, the appropriate dosage forms are a nasal spray or dry powder. Thedosage of the agents for intranasal administration is the amountdescribed above for IM administration. When administered by nasalaerosol or inhalation, these compositions may be prepared according totechniques well known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents.

The agents can be administered intrathecally. When given by this route,the appropriate dosage form can be a parenteral dosage form. The dosageof the compounds for intrathecal administration is the amount describedabove for IM administration.

The agents can be administered topically. When given by this route, theappropriate dosage form is a cream, ointment, or patch. Whenadministered topically, an illustrative dosage is from about 0.5 mg/dayto about 200 mg/day. Because the amount that can be delivered by a patchis limited, two or more patches may be used.

The agents can be administered rectally by suppository. Whenadministered by suppository, an illustrative therapeutically effectiveamount may range from about 0.5 mg to about 500 mg. When rectallyadministered in the form of suppositories, these compositions may beprepared by mixing the drug with a suitable non-irritating excipient,such as cocoa butter, synthetic glyceride esters of polyethyleneglycols, which are solid at ordinary temperatures, but liquefy and/ordissolve in the rectal cavity to release the drug.

In some cases, it may be desirable to administer a compound directly tothe airways of the patient, using devices such as, but not limited to,metered dose inhalers, breath-operated inhalers, multidose dry-powderinhalers, pumps, squeeze-actuated nebulized spray dispensers, aerosoldispensers, and aerosol nebulizers. For administration by intranasal orintrabronchial inhalation, the compounds of the present teachings can beformulated into a liquid composition, a solid composition, or an aerosolcomposition. The liquid composition can include, by way of illustration,one or more compound dissolved, partially dissolved, or suspended in oneor more pharmaceutically acceptable solvents and can be administered by,for example, a pump or a squeeze-actuated nebulized spray dispenser. Thesolvents can be, for example, isotonic saline, PBS, or bacteriostaticwater. In such embodiments, the pH of the pharmaceutical composition tobe administered may be from about 5.0 to about 7.0. The solidcomposition can be, by way of illustration, a powder preparationincluding one or more compound or salt, ester, solvate, hydrate, orprodrug thereof described above intermixed with lactose or other inertpowders that are acceptable for intrabronchial use, and can beadministered by, for example, an aerosol dispenser or a device thatbreaks or punctures a capsule encasing the solid composition anddelivers the solid composition for inhalation, i.e., blister packs. Insome embodiments, inhaled compositions encompassed by the invention mayinclude one or more additional inhaled therapeutic compounds. Forexample, in certain embodiments, the inhaled compositions may include acompound disclosed herein, and one or more additional therapeuticcompound such as, for example, bronchodilators such as β2 agonists(SABA/LABA) such as salmeterol, terbutaline, salbutamol, levosalbutamol,pirbuterol, bambuterol, fenoterol, metalproterenol, and formoterol,other bronchodilators such as epinephrine, racemic epinephrine,ephedrine, clenbuterol, indacaterol, vilanterol, and theophylline;corticosteroids such as beclomethasone, budesonide, ciclesonide,flunisolide, fluticasone and triamcinolone, anticholinergics such asipratropium and tiotropium, anti-inflammatories such as cromolyn andnedocromil, and the like and various combinations thereof. The aerosolcomposition can include, by way of illustration, one or more compound orsalt, ester, solvate, hydrate, or prodrug thereof described above,propellants, surfactants, and co-solvents, and can be administered by,for example, a metered device. The propellants can be achlorofluorocarbon (CFC), a hydrofluoroalkane (HFA), or otherpropellants that are physiologically and environmentally acceptable. Thedosage of the compounds of the invention administered directly to therespiratory system of the patient by, for example, inhalation may besimilar to the dosage administered systemically.

In certain embodiments, the compounds or compositions disclosed hereinmay be administered via intratracheal delivery, with or withoutco-administration of a bronchodilator such as β2 agonists (SABA/LABA)such as salmeterol, terbutaline, salbutamol, levosalbutamol, pirbuterol,bambuterol, fenoterol, metalproterenol, and formoterol, otherbronchodilators such as epinephrine, racemic epinephrine, ephedrine,clenbuterol, indacaterol, vilanterol, and theophyllines; corticosteroidssuch as beclomethasone, budesonide, ciclesonide, flunisolide,fluticasone and triamcinolone, anticholinergics such as ipratropium andtiotropium, anti-inflammatories such as cromolyn and nedocromil, and thelike and various combinations thereof.

In certain embodiments, the alginoketals may be co-administered withanother active agent. In certain embodiments, the active agent(particularly a therapeutically active agent) is included in a gel. Thegel may respond to external stimulus (e.g., physiological conditions)such as changes in ion concentration, pH, temperature, glucose, shearstress, or a combination thereof. Illustrative gels include hydrogelsinclude polyacrylamide (e.g., poly-N-isopropylacrylamide), siliconhydrogels like those used in contact lenses, polyethyleneoxide/polypropylene oxide or combinations of the two (e.g., Pluronicshydrogel or Tectronics hydrogel), butyl methacrylate, polyethyleneglycol diacrylate, polyethylene glycol of varying molecular weights,polyacrylic acid, poly methacrylic acid, poly lactic acid,poly(tetramethyleneether glycol), poly(N,N′-diethylaminoethylmethacrylate), methyl methacrylate, andN,N′-dimethylaminoethylmethacrylate. In certain embodiments, thehydrogel is a thermoresponsive hydrogel. Illustrative thermoresponsivegels include poly(N-alkylacrylamides), poly(N-vinyl caprolactam),poly(N-ethyl oxazoline), poly(methyl vinyl ether), poly(acrylicacid-co-acrylamide), elastin-like oligo- and polypeptides.

In certain embodiments, the thermoresponsive hydrogel has a lowercritical solution temperature (LCST) below body temperature. Thethermoresponsive hydrogel remains fluid below physiological temperature(e.g., 37° C. for humans) or at or below room temperature (e.g., 25°C.), solidify (into a hydrogel) at physiological temperature, and arebiocompatible. For example, the thermoresponsive hydrogel may be a clearliquid at a temperature below 34° C. which reversibly solidifies into agelled composition at a temperature above 34° C. Generally, theLCST-based phase transition occurs upon warming in situ as a result ofentropically-driven dehydration of polymer components, leading topolymer collapse. Various naturally derived and synthetic polymersexhibiting this behavior may be utilized. Natural polymers includeelastin-like peptides and polysaccharides derivatives, while notablesynthetic polymers include those based on poly(n-isopropyl acrylamide)(PNIPAAm), poly(N,N-dimethylacrylamide-co-N-phenylacrylamide),poly(glycidyl methacrylate-co-N-isopropylacrylamide), poly(ethyleneoxide)-b-poly(propylene oxide)-b-poly(ethylene oxide), poly(ethyleneglycol)-polyester copolymer, poly(ethylene glycol)-poly(serinolhexamethylene urethane), and amphiphilic block copolymers. The structureof PNIPAAm, containing both hydrophilic amide bonds and hydrophobicisopropyl groups, leads to a sharp phase transition at the LCST. Studiessuggest that the average number of hydrating water molecules per NIPAAmgroup falls from 11 to about 2 upon the hydrophobic collapse above theLCST (32-34° C.). In certain embodiments, the amphiphilic blockcopolymer comprises a hydrophilic component selected from poly ethyleneoxide (PEO), poly vinyl alcohol (PVA), poly glycolic acid (PGA), poly(N-isopropylacrylamide), poly(acrylic acid) (PAA), poly vinylpyrrolidone (PVP) or mixtures thereof, and a hydrophobic componentselected from polypropylene oxide (PPO), poly (lactic acid) (PLA), poly(lactic acid co glycolic acid) (PLGA), poly (.beta.-benzoyl L-aspartate)(PBLA), poly (.gamma.-benzyl-L-glutamate) (PBLG), poly (aspartic acid),poly (L-lysine), poly(spermine), poly (caprolactone) or mixturesthereof. Examples of such amphiphilic block copolymers include(PEO)(PPO)(PEO) block copolymers (PEO/PPO), and poly (lactic acid coglycolic acid) block copolymers (PLGA), such as (PEO)(PLGA)(PEO) blockcopolymers.

In certain embodiments, the hydrogel is non-biodegradable (e.g.,PNIPAAm). In other embodiments, the hydrogel is biodegradable. Forexample, biodegradable NIPAAm-based polymers can be made by conjugatingthe PNIPAAm with natural biodegradable segments such as MMP-susceptiblepeptide, gelatin, collagen, hyaluronic acid and dextran. Copolymersformed from NIPAAm and monomers with degradable side chains compriseanother category of NIPAAm-based bioabsorbable, thermoresponsivehydrogels. Hydrolytic removal of hydrophobic side chains increases thehydrophilicity of the copolymer, raising the LCST above body temperatureand making the polymer backbone soluble. Due to the relative simplicityof the synthetic process, the most investigated biodegradable monomershave been HEMA-based monomers, such as 2-hydroxyethylmethacrylate-polylactide (HEMA-PLA)(Lee, B. H.; et al. Macromol. Biosci.2005, 5, 629-635; and Guan, J., et al. Biomacromolecules 2008, 9,1283-92), 2-hydroxyethyl methacrylate-polycaprolactone (HEMA-PCL) (Wang,T., et al. Eur. J. Heart Fail 2009, 11, 14-19 and Wu, D., et al. ACSAppl. Mater. Interf. 2009, 2, 312-327) and 2-hydroxyethylmethacrylate-polytrimethylene carbonate (HEMA-PTMC) (Fujimoto, K. L., etal. Biomaterials 2009, 30, 4357-4368 and Wang, F., et al. Acta Biomater.2009, 5, 2901). However, the backbone remnant following hydrolysis,HEMA, presents hydroxyethyl side groups (—CH.sub.2CH.sub.2-OH), whichhave a relatively limited effect on remnant polymer hydrophilicity (Cui,Z., et al. Biomacromolecules 2007, 8, 1280-1286). In previous studies,such hydrogels have been found to be either partially bioabsorbable (Wu,D., et al. ACS Appl. Mater. Interf. 2009, 2, 312-327) or completelybioabsorbable, but have required the inclusion of considerablyhydrophilic co-monomers such as acrylic acid (AAc) in the hydrogelsynthesis (Fujimoto, K. L.; et al. Biomaterials 2009, 30, 4357-4368;Wang, F., et al. Acta Biomater. 2009, 5, 2901; and Guan, J., et al.Biomacromolecules 2008, 9, 1283-92).

In a further embodiment, the thermoresponsive hydrogel degrades anddissolves at physiological conditions in a time-dependent manner. Thecopolymer and its degradation products typically are biocompatible.According to one embodiment, the copolymer consists essentially ofN-isopropylacrylamide (NIPAAm) residues (a residue is a monomerincorporated into a polymer), hydroxyethyl methacrylate (HEMA) residuesand methacrylate-polylactide (MAPLA) macromer residues as disclosed inU.S. Patent Publ. 2012/0156176, which is incorporated herein byreference. Alternately, the copolymer consists essentially ofN-isopropylacrylamide residues, acrylic acid (AAc) residues, andhydroxyethyl methacrylate-poly(trimethylene carbonate) (HEMAPTMC)macromer residues as disclosed in U.S. Patent Publ. 2012/0156176, whichis incorporated herein by reference.

Additional biodegradable hydrogels include, but are not limited to,albumin, heparin, poly(hydroxyethylmethacrylate), fibrin,carboxymethylcellulose, hydroxypropylmethyl cellulose, lectin,polypeptides, agarose, amylopectin, carrageenan, chitin, chondroitin,lignin, hylan, α-methyl galactoside, pectin, starch, and sucrose.

The hydrogel may be made from a combination or mixture of any of thehydrogels disclosed herein.

The base precursor (e.g., a prepolymer, oligomer and/or monomer) for thegel, cross linkers, and initiators are mixed together and allowed topolymerize for a predefined period of time (from 1 h to 24 h typically)to form the gel. The gel is then washed to remove any excess initiatoror unreacted materials. The gel at this stage is a liquid (e.g., in theform of an aqueous solution) at room temperature until it is ready foruse. The active agent can be added in before, after, or during thepolymerization of the gel to form a suspension of solid active agent inthe gel.

It should be apparent to one skilled in the art that the exact dosageand frequency of administration will depend on the particular agentsadministered, the particular condition being treated, the severity ofthe condition being treated, the age, weight, general physical conditionof the particular subject, and other medication the individual may betaking as is well known to administering physicians or other clinicians.

EXAMPLES

Generation of Alginoketals

A 1 mg/ml alginic acid (MP Biomedical) solution was generated in DI H₂O.One (1) molar equivalent of 2,2-Bis(aminoethoxy)propane (Sigma Aldrich)was added with 50 mole excess of Sulfo-NHS (Fisher Scientific) and EDC(Fisher Scientific). The reaction was continued for 16 h at roomtemperature under stirring. The reaction product was then dialyzed using500 Da membrane (Spectrum Labs) for 16 h in DI H₂O to remove theunreacted EDC, sulfo-NHS and 2,2-Bis(aminoethoxy)propane. The dialyzedproduct, alginoketal was then lyophilized and analyzed using 1H NMR inD₂O at 90° C. (Bruker Avance III 400 MHz).

Generation of Alginoketal Particles

A 10 mg/mL solution of alginoketal in DI H₂O was generated by vortexingvigorously and used as the water phase along with 1.25 mL of 30%tween80. Span 80 (1.25 mL; Fisher Scientific) was dissolved in 75 mL ofoctane (Fisher Scientific), and used as the oil phase. In order togenerate protein encapsulated alginoketals, 10 mg of chicken egg whitealbumin (OVA—Sigma Aldrich) was added to the alginoketal solution andvortexed further for thorough mixing. This solution of alginoketalseither containing protein or not containing any protein was then addedto the oil phase under stirring at 10,000 rpm (Silverson L4RT-A) andallowed to mix for 3 min. DI H₂O or 700 min CaCl₂ solution or Copper(II) sulfate (70 mL; Fisher Scientific) solution made in DI H₂O was thenadded under stirring at 10,000 rpm and allowed to mix for 3 min.2-propanol (100 mL; Fisher Scientific) was then added to the mixture inorder to cure the particles, and allowed to mix for 3 min. The particlesobtained were centrifuged (Eppendorf) at 2000×Gs for 5 min and thesupernatant was discarded. The particles were then re-suspended in2-propanol, incubated at room temperature for 5 min and then centrifugedagain at 2000×Gs for 5 min. The particles were then re-suspended in 1×phosphate buffered saline (PBS—Fisher Scientific) and then centrifugedagain at 2000×Gs for 5 min. The alginoketal particles were thenlyophilized and used for further experiments. Alginate particles weremade using the same procedure as used for alginoketals.

Size of Alginoketal Particles

Size of alginoketal-Cu and alginoketal-Ca particles was determined usingdynamic light scattering (Malvern Instruments Inc., Zetasizer) and foundto be 800 nm and 1000 nm respectively. Qualitative analysis of theparticle size and shape was performed using scanning electron microscopy(JSM-6330F; JEOL).

pH Degradation of Alginoketals

OVA encapsulated alginoketal particles (1 mg) were incubated in 1×PBS(pH 7.4) for 6 days at 37° C. under circular motion for thorough mixing.The particles were centrifuged at 5,000×Gs for 5 minutes every day andthe supernatant was analyzed for the presence of OVA using BCA assay kit(Fisher Scientific). In order to assess the degradation of particles atendosomal pH, 3.33 mg of alginoketal particles were re-suspended in pH5.6 PBS buffer for 4 hr. The particles were centrifuged at 5,000×Gs for5 minutes, after 30 min, 1 hr, 2 hr, 3 hr and 4 hr and the supernatantwas analyzed for the presence of OVA using BCA assay kit (FisherScientific). The total amount of OVA encapsulated in the alginoketalparticles was analyzed by dissolving the 1 mg of alginoketal particlesin 500 mM EDTA solution in 1×PBS and 100 μL of 1N HCl for 30 min. Theamount of OVA in the solution was analyzed using BCA assay kit.

Cell Culture

M05 melanoma cells were cultured in M05 cell media consisting of 1 mg/mLantibiotic G418 (Sigma Aldrich) and 10% fetal bovine serum (FBS—FisherScientific), in complete RPMI (Fisher Scientific). M05 cells werepassaged every 3^(rd) day using 1× trypsin (Fisher Scientific) andsecond passage cells were used for all the experiments. HEK293 cells andHeLa cells were cultured in HeLa cell media consisting of 1% penicillinstreptomycin, 10% fetal bovine serum in DMEM/F12 (Fisher Scientific),and second passage cells were used for the experiments. HUVEC cells werecultured in 5 ng/mL EGF, 1% penicillin streptomycin, 10% fetal bovineserum in RPMI media, and second passage cells were used for theexperiments. MSC cells were cultured in 1% penicillin streptomycin, 1%anti-anti and 10% fetal bovine serum in α-MEM media and 1^(st) passagewas used for the experiments.

Endosome Disruption by Alginoketal Particles

M05 cells were seeded in the wells of the 96-well plate (FisherScientific) at 10⁴ cells per well for 16 h. alginoketal-Ca (100 μg/mL)or alginate particles (100 μg/mL) were incubated with 4 μM calcein(Sigma-Aldrich) with M05 cells for 15 min. Calcein is a membraneimpermeable dye and has been used for evaluating membrane integrity.Calcein at 4 μM without the addition of particles was utilized ascontrol. The cells were washed 3 times with 200 μL 1×PBS and imaged witha fluorescent microscope (Nikon). Images were obtained via bright fieldand FITC filters of the same field using a 20× objective and overlappedimages were generated using ImageJ software.

Depletion of Superoxides Generated Via Fenton's Reaction byAlginoketal-Cu

The ability of alginoketal-Cu to deplete the superoxides generated wereassessed by adding 50 μL of 100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL,6.25 μg/mL, 3.125 μg/mL, 1.5625 μg/mL alginoketal-Cu or alginoketal-Cato 10 μL of 0.1 mM FeCl₃ (Sigma Aldrich) made in DI H₂O to the wells ofa 96 well plate. Stabilized tetramethylbenzidine substrate (50 μL; Colorreagent B, R&D systems) was added immediately and the absorbance wasmeasured using the plate reader (Molecular Devices, SpectraMax M5) at650 nm.

Generation of Hydrogen Peroxide by Alginoketal-Cu

The ability of alginoketal-Cu to generate hydrogen peroxide was assessedby incubating 100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL,3.125 μg/mL, 1.5625 μg/mL alginoketal-Cu or alginoketal-Ca with 1 mM KO₂(Sigma Aldrich) for 1 h. Stabilized tetramethylbenzidine substrate (50μL; Color reagent B, R&D systems) and 1 ng/mL horseradish peroxidase(Sigma Aldrich) was then added to assess the levels of remainingsuperoxides and the absorbance was measured using the plate reader(Molecular Devices, SpectraMax M5) at 650 nm after 30 min of incubation.

Cell Death Using Alginoketal-Cu

HeLa, HEK293, M05, MSC and HUVEC cells (10⁴) were seeded in the wells ofa 96-well plate for 16 h. Alginoketal-Cu or alginoketal-Ca (100 μg/mL)or 1×PBS was added to the cells and incubated for 16 h. Live/dead assaywas then performed using live/dead assay kit (Life Technologies) andpercentage of live cells were determined.

The effect of different concentrations of alginoketal-Cu on M05 celldeath was also determined. M05 cells (10⁴) were seeded in the wells of a96 well plate for 16 h. Alginoketal-Cu was re-suspended in M05 cellmedia, and added to a final concentration of 100 μg/mL, 50 μg/mL, 25μg/mL or 10 μg/mL of alginoketal-Cu for 16 h. Alginoketal-Ca (100 μg/mL)and cells treated without any particles, were utilized as negativecontrols. Cells were then washed twice with 1×PBS, and live assay wasperformed using live/dead assay kit (Life Technologies). The percentageof live cells was determined by normalizing all the conditions with thenegative control of no particle treatment control.

Intracellular Hydrogen Peroxide Levels Generated by Alginoketal-Cu

In order to determine intracellular hydrogen peroxide levels in M05cells, dihydrodichlorofluorescein diacetate (DCF, Sigma Aldrich) wasutilized. M05 cells (10⁴) were seeded in the wells of the 96 well platefor 16 h. Alginoketal-Cu (100 μg/mL, 50 μg/mL, 25 μg/mL or 10 μg/mL)were added to the cells for 1 h. Alginoketal-Ca (100 μg/mL) and cellstreated without any particles, were utilized as negative controls. Thecells were washed twice with 1×PBS and 25 μM DCF made in dimethylsulfoxide (DMSO—Fisher Scientific) was added, in order to get a finalconcentration of 5 μM DCF in the cell culture. The cells were incubatedwith DCF for 10 min at 37° C., and the fluorescence was determined atexcitation/emission 488/540 nm using the plate reader. In a separate setof experiments, the same procedure was performed for DCF staining, andthe cells were imaged using the FITC filter via a fluorescent microscopeat 10× objective.

Statistical Analysis

Statistical analyses were performed using a student t-test and p-valuesfor each experiment were determined.

FIGS. 6A-6D. Stapled Acid-Sensitive Endosome Disrupting AlginatesModulate Survival in Mice and Tumor Growth. (FIG. 6A)

“Soluble treatment”—Soluble delivery of antibody against PD-L1(programmed death ligand 1), soluble delivery of 1-MT (1 methyltryptophan), soluble delivery of OVA (chicken egg white albumin) andsoluble delivery of poly I:C. These soluble delivery pertains toinjection of these components.

“Anti-PD-L1+OVA-poly(I:C)”—Antibody against PD-L1 incorporated in athermoresponsive gel called poly(ethylene glycol)-poly(serinolhexamethylene urethane), however other thermoresponsive material can beutilized+OVA and poly(I:C) encapsulated in alginoketal-Ca particles. Theactive agent-loaded, thermoresponsive gel and alginoketal-Ca particleswere mixed together and injected in mice.

“OVA-poly(I:C)”—OVA and poly(I:C) encapsulated in alginoketal-Caparticles. Alginoketal-Ca particles were injected in mice.

“Anti-PD-L1+1-MT”—Antibody against PD-L1 incorporated in athermoresponsive gel called poly(ethylene glycol)-poly(serinolhexamethylene urethane), however other thermoresponsive material can beutilized +1-MT encapsulated in alginoketal-Ca particles. The activeagent-loaded thermoresponsive gel and alginoketal-Ca particles weremixed together and injected in mice.

“Anti-PD-L1+1-MT+OVA-poly(I:C)”—Antibody against PD-L1 incorporated in athermoresponsive gel called poly(ethylene glycol)-poly(serinolhexamethylene urethane), however other thermoresponsive material or nothermoresponsive gel can be utilized +OVA and poly(I:C) encapsulated inalginoketal-Ca particles +1-MT encapsulated in alginoketal-Ca particles.The active agent-loaded thermoresponsive gel and alginoketal-Caparticles were mixed together and injected in mice.

Alginoketal particles were generated using an inverse emulsion method.Alginoketal particles were generated with tween 80 (1.25 mL of 30% v/vin DI H₂O—Fisher Scientific), Span 80 (1.25 mL; Fisher Scientific,Pittsburgh, Pa.), 5 mL of 10 mg/mL solution of alginoketals in DI H₂Oand 75 mL of iso-octane (Fisher Scientific, Pittsburgh, Pa.). In orderto generate OVA-poly(I:C) or 1-MT encapsulated alginoketal particles, 10mg of chicken egg white albumin (OVA—Sigma Aldrich, St. Louis, Mo.)+10mg of poly(I:C) (Sigma Aldrich, St. Louis, Mo.) or 50 mg of 1-MT wereadded to the alginoketal solution and vortexed further for thoroughmixing. This solution of alginoketals along with tween 80 were thenadded to the iso-octane solution under stirring at 10,000 rpm (SilversonL4RT-A, East Longmeadow, Mass.) and allowed to mix for 3 min. Next, 700mM CaCl₂ solution (70 mL; Fisher Scientific, Pittsburgh, Pa.) solutionmade in DI H₂O was added under stirring at 10,000 rpm and allowed to mixfor 3 min. 2-propanol (100 mL; Fisher Scientific, Pittsburgh, Pa.) wasthen added to the mixture in order to cure the particles, and allowed tomix for 3 min. The particles obtained were centrifuged at 1800×Gs for 1min and the supernatant was discarded. The particles were thenre-suspended in 2-propanol, incubated at room temperature for 5 min andthen centrifuged again. The alginoketal particles were then lyophilizedand used for further experiments.

Results

Alginoketals were generated by conjugating naturally occurring alginicacid with bis-amino ketals, via a straight-forward amide formingreaction, using 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC) and sulfo-N-hydroxysulfosuccinimide (sulfo-NHS)(FIG. 2A). The generation of Alginoketals was confirmed using 1H NMR(FIG. 2B) and FTIR (FIG. 2C), where the formation of the amide bond wasconfirmed by the shift in the carboxylic acid peak from 1595 in alginateto 1620 in alginoketals. Nanoparticles were generated from Alginoketalsusing inverse emulsions, and Ca or Cu was used as a binding agent.Although, the particles were poly-disperse as seen in the scanningelectron microscopy and dynamic light scattering experiments, allparticle populations measured remained in the endocytosable size range(FIG. 3A,B).

In order to deliver divalent cations to the cytosol, an endocytosableparticle should be capable of disrupting endosomes (pH-5). Therefore,alginoketals were generated using ketal linkages that are known tohydrolyze at pH 5. Hydrolysis of the ketals then can induce endosomedisruption, and release the divalent cations and encapsulated materialsin the cytosol of the cells. The effect of pH on Alginoketals was testedby encapsulating a model protein chicken egg white ovalbumin (OVA) anddetermining its release kinetics at endosomal pH 5.6 and physiologicalpH 7.4. We observed that at pH 5.6 Alginoketal particles hydrolyzed andreleased 100% of the encapsulated OVA within 2 hr (FIG. 3D), whereas atpH 7.4, only 20% of the OVA was released after 6 days (FIG. 3C).

The endosome disrupting ability of the Alginoketals was tested byincubating Alginoketal particles and membrane impermeable calcein dyewith M05 (B16F10-OVA expressing) cell lines. Alginoketals appear capableof delivering calcein to the cytosol of M05 cells within 15 min ofco-incubation (FIG. 4A), whereas poly (lactide-co-glycolide) particlesgenerated via water in oil emulsion were not able to deliver calcein tothe cytosol even after 1 h of incubation (FIG. 4B).

In order to demonstrate that Alginoketal-Cu can act as the superoxidedismutase mimic and scavenge superoxide radicals, we incubatedAlginoketal-Cu with superoxide radicals generating FeCl₃. Alginoketal-Cu(50 μL) at various concentrations were added to 50 μL of 0.1 mM FeCl₃and 50 μL tetramethylbenzidine (TMB) substrate. The change in absorbanceat 650 nm was measured immediately and lower levels of absorbancerepresented scavenging of the superoxide radicals (FIG. 5A). It wasobserved that Alginoketal-Cu were able to scavenge the radicalsgenerated by FeCl₃ in a dose dependent manner (FIG. 5B). In order tofurther elucidate the role of Alginoketal-Cu as a superoxide dismutasemimic, we determined if Alginoketal-Cu could convert superoxides intohydrogen peroxide. Excess cytosolic Cu(II) is known to induce theproduction of hydrogen peroxide, via Cu (II) to Cu (I) reactions in thepresence of superoxide (FIG. 5C), which then leads to cell death (12,18). The ability of Alginoketal-Cu to produce hydrogen peroxide in thepresence of superoxide was assessed by incubating varying concentrationsof Alginoketal-Cu with 1 mM KO₂ for 1 h. Tetramethylbenzidine andhorseradish peroxidase were then added in order to determine the levelsof hydrogen peroxide generated by Alginoketal-Cu. FIG. 5D demonstratesthat the absorbance at 650 nm corresponding to the level of hydrogenperoxide is directly correlated with the amount of Alginoketal-Cu, whichshows that Alginoketal-Cu were able to induce the production of hydrogenperoxide in a dose dependent manner.

In order to demonstrate that Alginoketals can deliver Cu(II) cations andmodulate the reactive oxygen species homeostasis in the cytosol of thecells, we generated Cu containing Alginoketals (Alginoketal-Cu) andincubated them with M05 melanoma cancer cells. Mammalian cells areexcellent at maintaining cytosolic Cu homeostasis via the expression ofcopper transporters and therefore, prevent cell death due to Cu toxicity(19, 20). In this report, we demonstrate that intracellular Cu levelscan be increased in M05 cancer cells using Alginoketal-Cu, which lead toincreased hydrogen peroxide levels and cell death. We also compared theeffects of Alginoketal-Cu on cancerous and non-cancerous cells.Alginoketal-Cu (100 μg/mL) were incubated with non-cancerous cells(MSCs, HEK293 and HUVECs) and cancerous cells (M05 and HeLa) overnightand their effect on cell death was observed. We observed thatAlginoketal-Cu preferentially induced cell death in cancerous cells ascompared to non-cancerous cell types (FIG. 5E). Different levels ofAlginoketal-Cu were incubated with the M05 cells for 16 h and percentagelive cells were determined. FIG. 5F shows that the Alginoketal-Cu appearto be capable of inducing cell death at 100 μg/mL, 50 μg/mL and 25μg/mL, whereas 10 μg/mL levels of Alginoketal-Cu does not induce celldeath. These data suggest that low levels of Cu support cell growthwhereas higher levels of Cu induce cell death. In order to determine ifCu induced the production of hydrogen peroxide, M05 cells were seeded in96-well plate and incubated with 100 μg/mL, 50 μg/mL, 25 μg/mL, and 10μg/mL levels of Alginoketal-Cu or 100 μg/mL of Alginoketal-Ca for 1 h.The amount of hydrogen peroxide generated, normalized to no treatmentcontrol shows that 100 μg/mL, 50 μg/mL and 25 μg/mL levels ofAlginoketal-Cu induced significantly higher amounts of intracellularhydrogen peroxide in cancer cells as compared to 100 μg/mLAlginoketal-Ca, which might lead to cell death (FIG. 5G). Theaccumulation of hydrogen peroxide by Alginoketal-Cu in M05 cells wasqualitatively assessed via fluorescent microscopy (FIG. 5H), which showsthat higher levels of alginoketal-Cu lead to induction of larger amountof intracellular accumulation of hydrogen peroxide in M05 cells.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention.

What is claimed is:
 1. An agent comprising: an algin crosslinked withacetal linkages, wherein the acetal linkages have a structure of—X¹-A-X²— wherein X¹ and X² are each independently —NH— or —O—, and Aincludes an acetal group; and at least one cation coupled to thecrosslinked algin; wherein A is:

 wherein R¹⁰ and R¹¹ are each independently an alkanediyl, and R¹² andR¹³ are each optionally substituted alkyl.
 2. An agent having astructure comprising:

wherein M is a cation; X¹ and X² are each independently —NH— or —O—, andwherein A is;

 wherein R¹⁰ and R¹¹ are each independently an alkanediyl, and R¹² andR¹³ are each optionally substituted alkyl; or

wherein M is a cation; X¹ and X² are each independently —NH— or —O—, andA is:

wherein R¹⁰ and R¹¹ are each independently an alkanediyl, and R¹² andR¹³ are each optionally substituted alkyl; or a combination of FormulaIV and Formula V.
 3. The agent of claim 1, wherein the cation isselected from copper, iron, calcium, zinc, magnesium, selenium,aluminum, manganese, barium, strontium or a combination thereof.
 4. Theagent of claim 1, wherein the acetal is derived from2,2-bis(aminoethoxy)propane, 1-(2-Hydroxyethyl)-4-piperidone ethyleneketal, 1-Amino-4-oxocyclohexanecarboxylic acid ethylene ketal, or4-Bromoacetophenone diethyl ketal.
 5. The agent of claim 1, wherein thealgin is derived from alginic acid.
 6. The agent of claim 1, wherein thecation is chelated to the algin.
 7. The agent of claim 1, wherein theagent includes at least two different types of cations.
 8. The agent ofclaim 1, wherein the agent is endocytosable.
 9. The agent of claim 1,wherein the acetal linkages or groups are pH cleavable at endosomal pH.10. A pharmaceutical composition comprising the agent of claim 1 and atleast one pharmaceutically acceptable additive.
 11. A pharmaceuticalcomposition comprising the agent of claim 1, and an active agent-loadedthermoresponsive gel.
 12. The pharmaceutical composition of claim 11,wherein the active agent is a therapeutically active agent.
 13. A methodfor increasing the intracellular level of at least one cation abovehomeostatic level in a cell, comprising contacting the cell with theagent of claim
 1. 14. A method comprising delivering at least one cationto the cytosol of a cell, comprising contacting the cell with the agentof claim
 1. 15. A method for increasing the intracellular level ofhydrogen peroxide above homeostatic level in a cell, comprisingcontacting the cell with the agent of claim
 1. 16. A method comprisingdelivering at least one cation to the cytosol of a prokaryotic oreukaryotic cell, comprising contacting the cell with the agent ofclaim
 1. 17. A method comprising delivering at least one cation to theextracellular space of a prokaryotic or eukaryotic cell, comprising notcontacting the cell with the agent of claim
 1. 18. The agent of claim 1,wherein the acetal is derived from 2,2-bis(aminoethoxy)propane.
 19. Theagent of claim 1, wherein the cation is calcium.
 20. The agent of claim19, wherein the acetal is derived from 2,2-bis(aminoethoxy)propane. 21.The agent of claim 1, wherein a single type of cation is coupled to thecrosslinked algin.
 22. A particle comprising the agent of claim
 1. 23. Acomposition comprising a plurality of the particles of claim
 22. 24. Thecomposition of claim 23, wherein the particles have an average particlesize of 1 nm to 10,000 μm.
 25. A pharmaceutical composition comprisingthe composition of claim
 23. 26. A method for increasing theintracellular level of at least one cation above homeostatic level in acell, comprising contacting the cell with the composition of claim 23.27. A method comprising delivering at least one cation to the cytosol ofa cell, comprising contacting the cell with the composition of claim 23.28. A method for increasing the intracellular level of hydrogen peroxideabove homeostatic level in a cell, comprising contacting the cell withthe composition of claim
 23. 29. The particle of claim 22, wherein theparticle encapsulates an immunotherapeutic agent, a cancer cytotoxicagent, or both an immunotherapeutic agent, a cancer cytotoxic agent. 30.The particle of claim 22, wherein the particle encapsulates a protein.